For the first time, researchers have demonstrated a highly significant relationship between a mother's exposure during pregnancy to phthalates—a family of compounds used widely in plastics and personal care products—and changes in the ways that baby boy's genitals develop.

In this paper, Swan et al. report that a sensitive index of demasculinization of the male reproductive tract, called the anogenital index (AGI), was significantly related to phthalate exposure:

Boys exposed to multiple phthalates simultaneously were also more likely to have smaller AGI scores.

Boys with lower AGIs had smaller penis volumes and were more likely to experience incomplete testicular descent.

Their study builds on a wealth of animal research showing that certain phthalates cause a cluster of effects-- called ‘the phthalate syndrome’-- which includes demasculinization of the male reproductive tract, increases in the likelihood of undescended testes, and lowered sperm counts and testicular tumors in adulthood.

Swan et al. also report that the levels of phthalates associated with significant AGI reductions are found in approximately one-quarter of Americans tested by the CDC for phthalate body burdens.

What did they do?

Swan et al. enlisted mothers from Los Angeles, CA, Minneapolis, MN, and Columbia, MO, who had participated in previous research conducted by the
The Study for Future Families Research Group. They were considered eligible if their pregnancies resulted in a live birth, the baby was 2-36 months old, lived within 50 miles of the clinic, and could attend one study visit. A total of 346 mothers participated in the study, and 176 male babies were born to this group of mothers.

Blood and urine samples were available for most of the women from the previous study. 85 prenatal urine samples from the mothers of boys were analyzed for 9 phthalate metabolites by scientists at the Centers for Disease Control and Prevention, who had no access to data on the subjects.

Pediatricians with no knowledge of the mother’s phthalate concentrations were trained to conduct a standard examination of the genitals of the infants and young children.

For boys, the pediatricians obtained measurements of anogenital distance and penis size, and noted whether the testicles were normal or incompletely descended, whether the scrotum was distinct from surrounding tissue or not, and whether the scrotum were small or not.

Out of the 176 boys, 134 (78%) were examined by the pediatricians. Boy babies from twin births were excluded from the study. In 2 cases, the mothers declined permission for the genital examination; the others were either too old or active to be reliably measured.

Since the boys were examined at different ages and varied in size, the researchers calculated an anogential index (AGI), defined as the anogenital distance (AGD) divided by the weight of the boy at examination. This allowed them to make comparisons across boys of different ages and weights.

Having collected both phthalate and morphological data, Swan et al. then carried out a series of statistical analyses to explore the relationships between phthalate concentrations measured in mothers before birth, and the various genital measurements on the baby boys.

They determined for each boy how their actual AGI compared to what was expected on the basis of their age.

Why anogenital distance?

Anogenital distance (AGD) is a measurement of the length of the perineum that toxicologists routinely use to determine the sex of newborn pups. It is easily measured and in rodents and humans and in both it is about twice as long in males as females.

Experiments have demonstrated that in rodent studies this distance is shortened when the mother is exposed to chemicals that are anti-androgenic, such as dibutyl phthalate (DBP) or benzylbutyl phthalate (BBzP).

In this study of baby boys, Swan et al. used anogenital distance as the indicator of demasculinization because it has proven to be an extremely reliable and sensitive measure of decreased androgen activity caused by phthalate exposure during fetal development in animal experiments.

One of its advantages is that it is a continuous variable, instead of something like a birth defect, which is often 'all or nothing.' With rare birth defects, very large sample sizes are required to detect statistically significant results. In contrast, changes in the average value of a continuous variable, like AGD, are more readily observed.

AGD is also important because it is an indicator of androgen action. The same decreases in androgen action that shorten the AGD in animals have a wide array of other effects, which have adverse impacts throughout the life of the male.

This allowed them to divide the boys into three groups for the purpose of comparison:

Boys whose AGI was beneath the 25 percentile for their age.

Boys with an intermediate AGI for their age

Boys with an AGI greater than the 75th percentile for their age

The researchers also examined the relationship between AGD and AGI compared to penile volume, testicular placement, and scrotal description (size and distinctness from surrounding tissue), and the proportion of boys in these three categories with normal testicular descent and normal scrotal development.

They also categorized phthalate levels. For each phthalate metabolite studied, boys were placed into one of 3 groups:

low (<25 th percentile)

intermediate (between 25 th and 75 th percentile)

high ( >75 th percentile)

Since concentrations of different phthalates tend to be correlated, and since the sample size was not large enough to reliably examine interactions between phthalates, combined phthalate exposure was examined for those phthalates that were most strongly associated with AGI, called AGI-associated phthalates. The sum of AGI-associated phthalates were categorized as follows:

Metabolites of two parent compounds, DnOP and DMP, were not related to AGI scores. Trends for two metabolites of the other parent compound, DEHP, were suggestive but not statistically significant.

Based on these correlations, Swan et al. identified the four metabolites with significant inverse correlations as "AGI-associated." They calculated odds ratios for having a short AGI (in the lowest 25% percentile) in relation to exposure category for each of the AGI-associated metabolites. For example, they found that mothers whose urine contained high concentrations of MBP prior to birth were 10 times more likely to have boys with a shorter than expected AGI compared to women with low concentrations (odds ratio = 10.2, 95% confidence interval = 2.5 – 42.4) and nearly 4 times as likely as women with intermediate concentrations (odds ratio = 3.8, 95% confidence interval = 1.2 – 12.3) .

Because these phthalates work as anti-androgens, Swan et al. wanted to test whether combinations of phthalates might be more powerful than phthalates by themselves. To do this, the created a 'summary phthalate score', which reflected simultaneous exposure to multiple phthalates. The higher the score, the more total phthalate exposure there was.
The summary phthalate score was directly related to the proportion of boys with short AGI (p = 0.001). "Of the 10 boys whose phthalate scores were high (score = 11-12), all but one had a short AGI. Conversely, of the 11 boys whose score were low (score = 0 or 1) only one had a short AGI."

They then calculated an odds ratio for having a short AGI for babies with high total exposure. Babies with high total exposure were ninety times more likely to have a short AGI, compared to babies with low total exposure. This is an extraordinarily large odds ratio. Because of the relatively small sample size, the 95% confidence limits for the estimate of this odds ratio is quite wide, from 4.88 to 1659. The lower limit of 4.88 itself is indicative of a very strong (almost 5-fold) increase in risk.

The graph below shows the impact of multiple phthalates.

As babies age, their AGI decreases, as indicated by the line marking the mean score. [Remember that AGI is anogenital distance corrected for weight.]

All babies from the high combined phthalate group fall beneath the mean AGI, and almost all beneath the 25th percentile. Almost all with low phthalate scores fall above the mean AGI. This pattern is highly significant statistically.

Low AGI scores were associated with other developmental differences: A boy with a short AGI had, on average, an AGI that was 19% shorter than expected based on his age and weight. Boys with short AGIs had an increased likelihood of incomplete testicular descent, a small and indistinct scrotum (p < 0.001), and smaller penis size (p< 0.001). Twenty percent of boys with a short AGI had incomplete testicular descent compared to 5.9% for boys with a long AGI.

No frank genital malformations or disease or other grossly abnormal measurement results were detected in any the baby boys. Overall, 86.6% of boys had normal testes (normally descended or normally retractile).

What does it mean?

This study is especially noteworthy for several reasons.

It is the first human study linking prenatal phthalate exposure to adverse effects on the male reproductive system. Boys with higher phthalate exposures had shorter anogenital distances, which in animals is a result of interference with androgens like testosterone. Androgen signaling is essential for proper development of the male reproductive tract. Boys with smaller AGI scores had smaller penises, were more likely to have incomplete testicular descent, and have a small and indistinct scrotum.

In rodents, interference with androgen signaling during male development has effects on a wide array of developmental processes, not just the genital tract. Long term changes in brain development and behavior are well-documented consequences.

The associations between prenatal phthalate exposure and decreased AGI were very large and highly statistically significant. By way of contrast, studies that find a two-fold increase in the probability that a certain exposure is associated with an adverse outcome are often considered noteworthy; this study found a 90-fold increase in the probability of adverse outcome (short AGI (AGD normalized by weight of the baby) when comparing high vs. low exposure to certain phthalates.

The results are highly plausible since they are consistent with results in animal studies and with known mechanisms of action of the phthalates studied.

In rodents, fetal exposure to phthalates causes what scientists have termed a 'phthalate syndrome.' It includes decreases in AGD, increased frequency of incomplete testicular descent and a birth defect of the penis called 'hypospadias,' increased risk of testicular cancer in adulthood, impaired sperm quality and other effects.

Scientists studying people have noted a pattern of male abnormalities called 'testicular dysgenesis syndrome.' It includes hypospadias, cryptorchidism, impaired sperm quality and testicular cancer. One leading hypothesis for the cause of TDS has been exposure to endocrine disrupting chemicals like phthalates.

Swan et al.'s results may provide the missing link between these two syndromes.

The study finds adverse effects on male babies at levels of phthalates typically encountered in the general population. The changes in AGI were observed at phthalate levels below those found in one-quarter of women in the United States, based on CDC's nation-wide sample.

The mothers in this study are typical of women in the general population, with no known occupational or otherwise unusual exposure to phthalates. Because exposure to phthalates is so ubiquitous, it is difficult if not impossible to find a “control” (unexposed) group of women. This study found that women in the top quartile of exposure were much more likely to have sons with subtle but measurable differences in their reproductive systems, compared to women in the lowest quartile of exposure. In fact, the levels of most of the AGI-associated phthalates were lower in this population compared to those obtained in a nationwide survey by the Centers for Disease Control (NHANES).